Compositions, method, and kits for treating pulmonary conditions

ABSTRACT

Compositions, methods, and kits useful for treating pulmonary conditions are provided herein. Such compositions can contain synergizing amounts of a non-specific phosphodiesterase inhibitor, such as a methylxanthine, in combination with leucine and/or a leucine metabolite, and resveratrol.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.14/339,096, filed Jul. 23, 2014, which application is acontinuation-in-part of PCT Application No. PCT/US14/11531, filed Jan.14, 2014, which claims benefit of priority to U.S. ProvisionalApplication No. 61/752,909, filed Jan. 15, 2013, each of which isincorporated herein by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Pulmonary diseases or conditions span a range of lung-related diseases,including asthma and chronic obstructive pulmonary disease (COPD), whichaffect millions of people throughout the world.

For example, over 300 million people worldwide suffering from asthma. Itis predicted that the prevalence will increase to about 400 million inthe next decade. Asthma is a chronic airway disorder identified byrecurrent wheeze and intermittent air flow limitation. It ischaracterized by airway inflammation, mucus hypersecretion, and airwayhyperresponsiveness (AHR). Studies have shown that these clinicalmanifestations are, at least in part, inflammatory responses mediated byT-helper type 2 (Th2) cells together with mast cells, B cells andeosinophils, as well as a number of inflammatory cytokines andchemokines.

Currently, there are three anti-inflammatory agents for controllingasthma, which include inhaled steroids, cysteinyl-leukotriene receptorantagonist and cromolyn. However, the therapeutic efficacies ofcysteinyl-leukotriene receptor antagonist and cromolyn are highlyvariable and may be limited to certain subgroup of patients. Inaddition, 5-10% of the asthmatics are not well-controlled by currentdrug treatment and they require oral steroids during exacerbation. Oralsteroid usage is commonly associated with a diversity of adverseeffects, most notably increases in appetite, stomach ulcers, difficultysleeping (insomnia), changes in mood and behavior, flushing (redness) ofthe face, and short-term weight gain due to increased water retention.If taken for long periods of time, steroid use may lead to glaucoma,cataracts, high-blood pressure, heart disease, diabetes mellitus,obesity, acid reflux/GERD, osteoporosis, myopathy, increase in certaintypes of infections, and cushing syndrome.

COPD is a medical condition that is generally considered to include oneor both of chronic bronchitis and emphysema. Chronic bronchitis ischaracterized by a persistent (such as more than one year) productivecough that is not due to a medically defined cause such as a microbialinfection or carcinoma. Emphysema is an abnormal permanent non-uniformenlargement of air spaces distal to the terminal bronchioles, includingdestruction of the walls of the air spaces. COPD is caused by noxiousparticles or gas, most commonly from tobacco smoking, which triggers anabnormal inflammatory response in the lung. The natural course of COPDis characterized by occasional sudden worsening of symptoms called acuteexacerbations, most of which are caused by infections or air pollution.

There is currently no cure for COPD and the only measures that have beenshown to reduce mortality are smoking cessation and supplemental oxygen.COPD is treated with bronchodilators such as beta-2 agonists and/oranticholinergics. Beta-2 agonist stimulate beta-2 receptors whileanticholinergics block stimulation from cholinergic nerves both aremedicines that relax smooth muscle around the airways, increasing airflow. While these agents can ameliorate certain symptoms to some degree,they are not effective to halt progression of COPD.

Methylxanthines (a class of derivatives of xanthine and alkaloids) haveoften been used as bronchodilators. Methylxanthines relax smooth muscle,stimulate the central nervous system, stimulate cardiac muscle, and acton the kidneys to promote diuresis. Their usefulness in promotingrelaxation of bronchial smooth muscle is of benefit in the management ofasthma.

Methylxanthine theophylline is an established medicament for therapy ofobstructive diseases of the respiratory tract. Theophylline is acompetitive but non-selective inhibitor of several types ofphosphodiesterases, the enzymes that degrade cAMP. Increasedconcentrations of cAMP may mediate the observed bronchodilation. Otherproposed mechanisms of action of theophylline include inhibition of therelease of intracellular calcium and competitive antagonism of thebronchoconstrictor adenosine.

Theophylline is a relatively low-cost treatment and can be administeredin a sustained-release preparation that gives a duration of around 12hours. However, theophylline has a number of side effects. The adversegastrointestinal effects of theophylline include nausea, vomiting,abdominal pain, cramping, and diarrhea. Adverse central nervous systemeffects include insomnia, headache, dizziness, nervousness, andseizures, which are often more severe in children. Seizures may occur asthe initial sign of theophylline toxicity without any other precedingsigns and symptoms. Increased tremor in the patient's dominant hand hasbeen reported. Cardiovascular and pulmonary adverse effects includetachycardia, arrhythmias, and tachypnea. Because of these toxicities,theophylline is often used as a second or third line asthma medication.

SUMMARY OF THE INVENTION

There remains a considerable need for low-cost therapy that caneffectively and safely treat pulmonary conditions. The present inventionaddresses this need and provides related advantages as well.

The subject application provides compositions, methods, and kits usefulfor treating pulmonary conditions, including but not limited to asthmaand chronic obstructive pulmonary disease. The subject compositions areparticularly effective in reducing expression and/or secretion of aninflammatory marker or increasing the expression and/or secretion of ananti-inflammatory marker associated with the inflammatory responseselicited during onset of asthma or chronic obstructive pulmonarydisease.

In one aspect of the invention, the subject composition comprisesleucine and/or one or more leucine metabolites selected from the groupconsisting of keto-isocaproic acid (KIC), alpha-hydroxy-isocaproic acid,and hydroxymethylbutyrate (HMB); and a methylxanthine, wherein thecomposition comprises at least about 250 mg of leucine and/or at leastabout 100 mg of the one or more leucine metabolites. In one aspect ofthe invention, the subject composition comprises leucine and/or one ormore leucine metabolites selected from the group consisting ofketo-isocaproic acid (KIC), alpha-hydroxy-isocaproic acid, andhydroxymethylbutyrate (HMB); and a methylxanthine, wherein thecomposition comprises at least about 250 mg of leucine and/or at leastabout 10 or 50 mg of the one or more leucine metabolites. Thecomposition can further comprise resveratrol.

In some embodiments, the composition comprises at least about 500 mg ofleucine. The composition can comprise between about 250-1500 mg ofleucine. The composition can comprise at least about 200 mg of leucinemetabolites. The composition can comprise between about 100-750 mg ofleucine metabolites. The composition can comprise between about 10-750or 50-750 mg of leucine metabolites.

In another embodiment, the methylxanthine is selected from the groupconsisting of theophylline and theobromine. The methylxanthine may bepresent in a sub-therapeutic amount. The composition may comprise atleast about 5 mg of theophylline. The composition can comprise betweenabout 1-100 or 5-50 mg of theophylline. The composition can comprise asub-therapeutic amount of theophylline.

In one embodiment, the composition comprises at least about 35 mg ofresveratrol. The composition can comprise between about 5-500 or 30-250mg of resveratrol.

In yet another embodiment, the composition is substantially free ofnon-branched amino acids. The amino acids in the composition can besubstantially free of non-branched amino acids. The percent ofnon-branched amino acids relative to total amino acids in thecomposition can be less than about 0.1, 1, or 10%.

In some embodiments, the composition is a unit dosage. The compositioncan be formulated for oral dosing, inhalation, or intravenous delivery.The composition can be formulated for sustained release over a period ofat least about 1, 4, 6, 12, 24, 36, or 48 hours. The sustained releaseformulations may maintain a desired circulating level of one or morecomponents of the composition over a period of at least about 1, 4, 6,12, 24, 36, or 48 hours. The sustained release formulations may effect acirculating level greater than a desired circulating level over a periodof at least about 4, 6, 12, 24, 36, or 48 hours. The desired circulatinglevel may be for any component of the compositions. The component may bea methylxanthine, such as theophylline. In some embodiments, thesustained release composition maintains the level of the methylxanthineat greater than about 1 μM for the specified time period. The sustainedrelease formulation can effect a circulating level of methylxanthine ina subject that is greater than about 1 μM for the time period. Thesustained release formulation can effect a circulating level ofmethylxanthine in a subject that is between about 1-10, 1-20, 1-30, or1-40 μM for the time period.

In another aspect of the invention, the subject composition comprises(a) leucine and/or one or more leucine metabolites selected from thegroup consisting of keto-isocaproic acid (KIC), alpha-hydroxy-isocaproicacid, and hydroxymethylbutyrate (HMB); and (b) a methylxanthine, whereinthe mass ratio of (a) to (b) is at least about 15, 25, 50, 75, or 100,and wherein the composition comprises at least about 5 mg of themethylxanthine. The composition can further comprise at least about 10mg of resveratrol. In some embodiments, the methylxanthine istheophylline or theobromine. The composition may be formulated as a unitdosage.

In one aspect of the invention, the subject composition comprises (a)leucine and/or one or more leucine metabolites selected from the groupconsisting of keto-isocaproic acid (KIC), alpha-hydroxy-isocaproic acid,and hydroxymethylbutyrate (HMB); and (b) a methylxanthine, wherein themolar ratio of (a) to (b) is at least about 400, 500, 750, or 1000, andwherein the composition comprises at least about 0.05, 0.1, 0.5, 1, or 2μg of the methylxanthine. The composition can comprise at least about0.05, 0.1, 0.5, 1, or 2 mg of the methylxanthine. The composition canfurther comprise at least about 15, 30, 50, 100, or 500 μg of leucine.The composition can further comprise at least about 5, 10, 15, 30, 50,100, or 500 mg of leucine. The composition can further comprise at leastabout 0.01, 0.05, 0.1, 0.5, or 1 μg of resveratrol. The composition canfurther comprise at least about 0.01, 0.05, 0.1, 0.5, or 1 mg ofresveratrol. In some embodiments, the methylxanthine is theophylline ortheobromine. The composition may be formulated as a unit dosage forinhalation. The composition may be packaged in an inhaler. The inhalercan comprise at least about 20, 50, 200, or 1000 unit dosages of thecomposition.

In yet another aspect of the invention, the subject compositioncomprises (a) leucine; and (b) a methylxanthine, wherein component (a)and (b) are present in an amount effective to achieve a circulatinglevel of about 0.3-2 mM leucine and about 0.5-10 μM methylxanthine in asubject. The amounts of (a) and (b) can be selected to induce acirculating level of about 0.7-2 mM leucine and about 0.7-3 μMmethylxanthine in a subject. The subject can be a human, a domesticatedanimal, or a farm animal.

In another aspect, the subject composition comprises (a) leucine; and(b) a methylxanthine, wherein component (a) and (b) are present in anamount that is effective in improving a pulmonary condition by reducingexpression level or secretion of one or more inflammatory markers in alung endothelial cell selected from the group consisting of NFκB,eotaxin, IL1-β, and IL6 or increasing expression level or secretion ofone or more anti-inflammatory markers in a lung endothelial cellselected from the group consisting of adiponectin receptor 1 andadiponectin receptor 2.

In still another aspect, the subject composition comprises (a) leucine;and (b) a methylxanthine, wherein component (a) and (b) are present inan amount that is effective in reducing expression level or secretion ofone or more inflammatory markers in a lung endothelial cell selectedfrom the group consisting of NFκB, eotaxin, IL1-β, and IL6, orincreasing expression level or secretion of one or moreanti-inflammatory markers in a lung endothelial cell selected from thegroup consisting of adiponectin receptor 1 and adiponectin receptor 2.The amount of (b) may be a sub-therapeutic amount. Alternatively, theamount of (b) may be less than an amount required to achieve acirculating level of about 40 μM.

The invention also provides for a method of treating pulmonaryconditions in a subject in need of treatment comprising administering tothe subject a composition of any one of the subject compositions.

In another aspect, the invention provides for a method of reducingexpression level or secretion of an inflammatory marker selected fromthe group consisting of NFκB, eotaxin, IL1-β, and IL6 or increasingexpression level or secretion of an anti-inflammatory marker selectedfrom the group consisting of adiponectin receptor 1 and adiponectinreceptor 2, comprising contacting a lung endothelial cell with acomposition of any one of the subject compositions described herein toeffect said reduction or increase in expression level or secretion ofsaid inflammatory marker or said anti-inflammatory marker. The subjectcan be a human, a domesticated animal, or a farm animal.

In yet another aspect, the invention provides for a kit comprising amulti-day supply of unit dosages of a subject composition describedherein, and instructions directing the administration of said multi-daysupply over a period of multiple days.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawing(s) of which:

FIG. 1 depicts the interactive effects of theophylline with leucine andresveratrol on NFκB protein expression in mouse lung endothelial cells.*p<0.02 vs. control.

FIG. 2 depicts the interactive effects of theophylline with leucine andresveratrol (200 nM) conditioned adipocyte media on NFκB proteinexpression in mouse lung endothelial cells.

FIG. 3 depicts the interactive effects of theophylline with leucine andresveratrol conditioned adipocyte media on phospho-NFκB proteinexpression in mouse lung endothelial cells.

FIG. 4 depicts the interactive effects of theophylline with leucine andresveratrol on IL1-β from mouse lung endothelial cells. *p<0.001 vs. allother treatments. TNFα used as a positive control to stimulate secretionof the cytokine; **indicates significant increase vs. all othertreatments (p<0.01).

FIG. 5 depicts the interactive effects of theophylline with leucine andresveratrol on eotaxin secretion from mouse lung endothelial cells.*p<0.05 vs. all other treatments. TNFα used as a positive control tostimulate secretion of the cytokine.

FIG. 6 depicts the interactive effects of theophylline with leucine andresveratrol conditioned adipocyte media on eotaxin secretion from mouselung endothelial cells.

FIG. 7 depicts the interactive effects of theophylline with leucine andresveratrol on cellular IL 6 content in mouse lung endothelial cells.*p<0.02 vs. control.

FIG. 8 depicts the interactive effects of theophylline with leucine andresveratrol on IL 6 secretion from mouse lung endothelial cells.*p<0.0001 vs. all other treatments. TNFα used as a positive control tostimulate secretion of the cytokine; **indicates significant increasevs. all other treatments.

FIG. 9 depicts the interactive effects of theophylline with leucine andresveratrol conditioned adipocyte media on IL 6 secretion from mouselung endothelial cells.

FIG. 10 depicts the interactive effects of theophylline with leucine andresveratrol on adiponectin receptor 1 protein expression in mouse lungendothelial cells.

FIG. 11 depicts the interactive effects of theophylline with leucine andresveratrol on adiponectin receptor 2 protein expression in mouse lungendothelial cells.

FIG. 12 depicts the interactive effects of theophylline with leucine andresveratrol conditioned adipocyte media on adiponectin receptor 2protein expression in mouse lung endothelial cells. *p=0.018 vs. allother treatments.

FIG. 13 depicts the interactive effects of theobromine with leucine andresveratrol conditioned adipocyte media on adiponectin receptor 1protein expression in mouse lung endothelial cells. *p=0.03 vs. allother treatments; **p=0.0005 vs. all other treatments.

FIG. 14 depicts the interactive effects of theobromine with leucine andresveratrol conditioned adipocyte media on adiponectin receptor proteinexpression in mouse lung endothelial cells. *p=0.0001 vs. all othertreatments.

FIG. 15 depicts the interactive effects of administration of leucine andtheophylline on eosinophil cell number in adult female Balb/c mice.

FIG. 16 depicts the interactive effects of administration of leucine andtheophylline on T-cell number in adult female Balb/c mice.

FIG. 17 depicts the interactive effects of administration of leucine andtheophylline on the T-cell percentage in adult female Balb/c mice.

DETAILED DESCRIPTION OF THE INVENTION

Several aspects of the invention are described below with reference toexample applications for illustration. It should be understood thatnumerous specific details, relationships, and methods are set forth toprovide a full understanding of the invention. One having ordinary skillin the relevant art, however, will readily recognize that the inventioncan be practiced without one or more of the specific details or withother methods. Unless stated otherwise, the present invention is notlimited by the illustrated ordering of acts or events, as some acts mayoccur in different orders and/or concurrently with other acts or events.Furthermore, not all illustrated acts or events are required toimplement a methodology in accordance with the present invention. Theconcentration of various components in the disclosed compositions areexemplary and not meant to be limited to the recited concentration perse.

As used herein, the term “subject” or “individual” includes mammals.Non-limiting examples of mammals include humans and mice, includingtransgenic and non-transgenic mice. The methods described herein can beuseful in both human therapeutics, pre-clinical, and veterinaryapplications. In some embodiments, the subject is a mammal, and in someembodiments, the subject is human. Other mammals include, and are notlimited to, apes, chimpanzees, orangutans, monkeys; domesticated animals(pets) such as dogs, cats, guinea pigs, hamsters, mice, rats, rabbits,and ferrets; domesticated farm animals such as cows, buffalo, bison,horses, donkey, swine, sheep, and goats; or exotic animals typicallyfound in zoos, such as bear, lions, tigers, panthers, elephants,hippopotamus, rhinoceros, giraffes, antelopes, sloth, gazelles, zebras,wildebeests, prairie dogs, koala bears, kangaroo, pandas, giant pandas,hyena, seals, sea lions, and elephant seals.

The terms “administer”, “administered”, “administers” and“administering” are defined as the providing a composition to a subjectvia a route known in the art, including but not limited to intravenous,intraarterial, oral, nasal, inhaled, parenteral, buccal, topical,transdermal, rectal, intramuscular, subcutaneous, intraosseous,transmucosal, or intraperitoneal routes of administration. In certainembodiments of the subject application, oral routes of administering acomposition may be preferred.

As used herein, “agent” or “biologically active agent” refers to abiological, pharmaceutical, or chemical compound or other moiety.Non-limiting examples include simple or complex organic or inorganicmolecule, a peptide, a protein, a peptide nucleic acid (PNA), anoligonucleotide (including e.g., aptomer and polynucleotides), anantibody, an antibody derivative, antibody fragment, a vitaminderivative, a carbohydrate, a toxin, or a chemotherapeutic compound.Various compounds can be synthesized, for example, small molecules andoligomers (e.g., oligopeptides and oligonucleotides), and syntheticorganic compounds based on various core structures. In addition, variousnatural sources can provide compounds for screening, such as plant oranimal extracts, and the like. A skilled artisan can readily recognizethat there is no limit as to the structural nature of the agents of thepresent invention.

The term “effective amount” or “therapeutically effective amount” refersto that amount of a compound described herein that is sufficient toeffect the intended application including but not limited to diseasetreatment, as defined below. The therapeutically effective amount mayvary depending upon the intended application (in vitro or in vivo), orthe subject and disease condition being treated, e.g., the weight andage of the subject, the severity of the disease condition, the manner ofadministration and the like, which can readily be determined by one ofordinary skill in the art. The term also applies to a dose that willinduce a particular response in target cells, e.g., reduction ofproliferation or down regulation of activity of a target protein. Thespecific dose will vary depending on the particular compounds chosen,the dosing regimen to be followed, whether it is administered incombination with other compounds, timing of administration, the tissueto which it is administered, and the physical delivery system in whichit is carried.

The term “isolated”, as applied to a component of a subject composition,such component including, for example, a methylxanthine PDE inhibitor(including but not limited to theophylline and theobromine), leucine andleucine metabolites (including HMB, KIC and alpha-hydroxy-isocaproicacid), and resveratrol, refers to a preparation of the substance devoidof at least some of the other components that may also be present wherethe substance or a similar substance naturally occurs or is initiallyobtained from. Leucine or its metabolite when used in the subjectcomposition is general in its free form and not as part of a polypeptideor a biomolecule. An isolated substance may be prepared by using apurification technique to enrich it from a source mixture. Enrichmentcan be measured on an absolute basis, such as weight per volume ofsolution, or it can be measured in relation to a second, potentiallyinterfering substance present in the source mixture. Increasingenrichment of the embodiments of this invention are increasingly morepreferred. Thus, for example, a 2-fold enrichment is preferred, 10-foldenrichment is more preferred, 100-fold enrichment is more preferred,1000-fold enrichment is even more preferred. A substance can also beprovided in an isolated state by a process of artificial assembly, suchas by chemical synthesis.

A “modulator” of a pathway refers to a substance or agent whichmodulates the activity or expression of one or more cellular proteinsmapped to the same specific signal transduction pathway. A modulator mayaugment or suppress the activity and/or expression level or pattern of asignaling molecule. A modulator can activate a component in a pathway bydirectly binding to the component. A modulator can also indirectlyactivate a component in a pathway by interacting with one or moreassociated components. The output of the pathway can be measured interms of the expression or activity level of proteins. The expressionlevel of a protein in a pathway can be reflected by levels ofcorresponding mRNA or related transcription factors as well as the levelof the protein in a subcellular location. For instance, certain proteinsare activated by translocating in or out of a specific subcellularcomponent, including but not limited to nucleus, mitochondria, endosome,lysosome or other membraneous structure of a cell. The output of thepathway can also be measured in terms of physiological effects, such asmitochondrial biogenesis, fatty acid oxidation, or glucose uptake.

An “activator” refers to a modulator that influences a pathway in amanner that increases the pathway output. Activation of a particulartarget may be direct (e.g. by interaction with the target) or indirect(e.g. by interaction with a protein upstream of the target in asignaling pathway including the target).

A “suppressor” can be a modulator that influences a pathway in a mannerthat decreases pathway output.

The term “substantially free”, as used herein, refers to compositionsthat have less than about 10%, less than about 5%, less than about 1%,less than about 0.5%, less than 0.1% or even less of a specifiedcomponent. For example a composition that is substantially free ofnon-branched chain amino acids may have less than about 1% of thenon-branched chain amino acid lysine. The percentage may be determinedas a percent of the total composition or a percent of a subset of thecomposition. For example, a composition that is substantially free ofnon-branched chain amino acids may have less than 1% of the non-branchedchain amino acids as a percent of the total composition, or as a percentof the amino acids in the composition. The percentages may be mass,molar, or volume percentages.

A “sub-therapeutic amount” of an agent, an activator or a therapy is anamount less than the effective amount of that agent, activator ortherapy for an intended application, but when combined with an effectiveor sub-therapeutic amount of another agent or therapy can produce adesired result, due to, for example, synergy in the resultingefficacious effects, and/or reduced side effects.

A “synergistic” or “synergizing” effect can be such that the one or moreeffects of the combination compositions are greater than the one or moreeffects of each component alone, or they can be greater than the sum ofthe one or more effects of each component alone. The synergistic effectcan be about, or greater than about 10, 20, 30, 50, 75, 100, 110, 120,150, 200, 250, 350, or 500% or even more than the effect on a subjectwith one of the components alone, or the additive effects of each of thecomponents when administered individually. The effect can be any of themeasurable effects described herein.

Compositions

The subject compositions comprise a combination of (i) a non-specificPDE inhibitor, such as a methylxanthine, and (ii) leucine and/or one ormore leucine metabolites. The compositions may further compriseresveratrol. The combination of these components can be useful fortreating pulmonary conditions, including but not limited to asthma andchronic obstructive pulmonary disease. The combination can beparticularly effective in reducing expression and/or secretion of aninflammatory marker or increasing expression and/or secretion of ananti-inflammatory marker associated with the inflammatory responseselicited during onset of asthma or chronic obstructive pulmonarydisease. In some embodiments, the components are formulated to provide asynergistic effect, including but not limited to reduction in dosingamounts leading to reduced side effects to the subject and/or reducedcost of treatment. In other embodiments, the synergistic effect canallow for results that are not achievable through any other conventionaltreatments.

In one embodiment, the subject composition comprises leucine and/or oneor more leucine metabolites selected from the group consisting ofketo-isocaproic acid (KIC), alpha-hydroxy-isocaproic acid, andhydroxymethylbutyrate (HMB); and a methylxanthine, wherein thecomposition comprises at least about 250 mg of leucine and/or at leastabout 100 mg of the one or more leucine metabolites.

In another embodiment, the subject composition comprises (a) leucineand/or one or more leucine metabolites selected from the groupconsisting of keto-isocaproic acid (KIC), alpha-hydroxy-isocaproic acid,and hydroxymethylbutyrate (HMB); and (b) a methylxanthine, wherein themass ratio of (a) to (b) is at least about 15, 25, 50, 75, or 100, andwherein the composition comprises at least about 5 mg of themethylxanthine. As described herein, a dosing of at least about 5 mg ofmethylxanthine can provide a sub-therapeutic dosing that can beeffective when combined with a sufficient mass ratio of leucine orleucine metabolite.

In one aspect of the invention, the subject composition comprises (a)leucine and/or one or more leucine metabolites selected from the groupconsisting of keto-isocaproic acid (KIC), alpha-hydroxy-isocaproic acid,and hydroxymethylbutyrate (HMB); and (b) a methylxanthine, wherein themolar ratio of (a) to (b) is at least about 400, 500, 750, or 1000, andwherein the composition comprises at least about 0.05, 0.1, 0.5, 1, or 2μg of the methylxanthine. The composition can comprise at least about0.05, 0.1, 0.5, 1, or 2 mg of the methylxanthine. The composition cancomprise at most about 0.001, 0.01, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 5, 10,or 20 grams of a methylxanthine, which may be theophylline and/ortheobromine. The composition can further comprise at least about 15, 30,50, 100, or 500 μg of leucine. The composition can further comprise atleast about 5, 10, 15, 30, 50, 100, or 500 mg of leucine. Thecomposition can comprise at most about 0.001, 0.01, 0.1, 0.5, 1, 1.5, 2,2.5, 3, 5, 10, or 20 grams of leucine and/or a leucine metabolite. Thecomposition can further comprise at least about 0.01, 0.05, 0.1, 0.5, or1 μg of resveratrol. The composition can further comprise at least about0.01, 0.05, 0.1, 0.5, or 1 mg of resveratrol. In some embodiments, themethylxanthine is theophylline or theobromine. The composition may beformulated as a unit dosage for inhalation.

In some embodiments, the composition is held within an inhaler designedto hold at least about 20, 50, 200, or 1000 unit dosages. The inhalercan comprise at least about 0.05, 0.1, 0.5, 1, or 2 μg of amethylxanthine per unit dose. The inhaler can comprise at least about15, 30, 50, 100, or 500 of leucine per unit dose. The inhaler cancomprise at least about 0.01, 0.05, 0.1, 0.5, or 1 μg of resveratrol perunit dose.

In yet another embodiment, the subject composition comprises (a)leucine; and (b) a methylxanthine, wherein component (a) and (b) arepresent in an amount effective to achieve a circulating level of about0.3-2 mM leucine and about 0.5-10 μM methylxanthine in a subject. Thesetargeted circulating levels correspond to treatment concentrationsdescribed herein (see Examples), which were shown to provide beneficialeffects on pulmonary conditions in a subject.

In still another embodiment, the subject composition comprises (a)leucine; and (b) a methylxanthine, wherein component (a) and (b) arepresent in an amount that is effective in reducing expression level orsecretion of one or more inflammatory markers in a lung endothelial cellselected from the group consisting of NFκB, eotaxin, IL1-β, and IL6, orin increase expression level or secretion of one or moreanti-inflammatory markers selected from the group consisting ofadiponectin receptor 1 and adiponectin receptor 2. The amount of (b) maybe a sub-therapeutic amount. Alternatively, the amount of (b) may beless than an amount required to achieve a circulating level of about 40μM. As described in the Examples, the combination of leucine and amethylxanthine can be combined to in amounts that have a beneficialeffect on reducing expression level or secretion of one or moreinflammatory markers in a lung endothelial cell such as NFκB, eotaxin,IL1-β, and IL6, or increasing the expression level or secretion of oneor more anti-inflammatory markers selected from the group consisting ofadiponectin receptor 1 and adiponectin receptor 2.

Phosphodiesterase Inhibitors

In some embodiments, the compositions can include a phosphodiesterase(PDE) inhibitor, such as a non-selective PDE inhibitor. PDE inhibitorscan be naturally occurring or non-naturally occurring (e.g.manufactured), and may be provided in the form of a natural sourcecomprising the PDE inhibitor, or an extract thereof (e.g. purified).Examples of non-selective PDE inhibitors include, but are not limitedto, caffeine, theophylline, theobromine, 3-isobutyl-1-methylxanthine(IBMX), pentoxifylline(3,7-dihydro-3,7-dimethyl-1-(5oxohexyl)-1H-purine-2,6-dione),aminophylline, paraxanthine, and salts, derivatives, metabolites,catabolites, anabolites, precursors, and analogs thereof. Non-limitingexamples of natural sources of PDE inhibitors include coffee, tea,guarana, yerba mate, cocoa, and chocolate (e.g. dark chocolate). Invarious embodiments, compositions are formulated such that they do notcontain (or exclude) one or more of the following ingredients: caffeine,green tea extract or extracts from guarana seed or guarana plants.Examples of phosphodiesterase inhibitors that may be used in a subjectcompositions are described in U.S. patent application Ser. No.13/549,381, filed Jul. 13, 2012, which is herein incorporated byreference in its entirety.

The PDE inhibitors may also be methylxanthines. Examples ofmethylxanthines include caffeine, ephedrine, oxtriphylline,aminophylline, paraxanthine, IBMX, pentoxifylline, theobromine, andtheophylline. Examples of aminophylline formulations includeaminophylline Boehringer (Boehringer Ingelheim GmbH). Examples ofephedrine formulations include Bronkaid® (Bayer AG), broncholate(Sanofi-Aventis), Primatene® (Wyeth), tedral SA®, and marax (PfizerInc). Examples of theophylline formulations include euphyllin (NycomedInternational Management GmbH), and theo-dur (Pfizer Inc, TevaPharmaceutical Industries Ltd). Examples of oxtriphylline formulationsinclude Choledyl SA (Pfizer Inc).

Leucine and Leucine Metabolites

The invention provides for compositions that include leucine and/orleucine metabolites. The leucine and/or leucine metabolites can be usedin free form. The term “free,” as used herein in reference to acomponent, indicates that the component is not incorporated into alarger molecular complex. For example a composition can include freeleucine that is not incorporated in a protein or freehydroxymethylbutyrate. The leucine can be L-leucine.

Without being limited to theory, ingestion of branched chain aminoacids, such as leucine, can stimulate sirtuin signaling, including Sirt1and Sirt3, as well as AMPK signaling, one or more of which can favorablymodulate inflammatory cytokine patterns. In some embodiments, any of thecompositions described herein can include salts, derivatives,metabolites, catabolites, anabolites, precursors, and analogs ofleucine. For example, the metabolites can include hydroxymethylbutyrate(HMB), keto-isocaproic acid (KIC), and keto isocaproate. The HMB can bein a variety of forms, including calcium 3-hydroxy-3-methylbutyratehydrate.

In certain embodiments of the invention, any of the compositionsdisclosed herein can be formulated such that they do not contain (orexclude) one or more amino acids selected from the group consisting oflysine, glutamate, proline, arginine, valine, isoleucine, aspartic acid,asparagine, glycine, threonine, serine, phenylalanine, tyrosine,histidine, alanine, tryptophan, methionine, glutamine, taurine,carnitine, cystine and cysteine.

In some embodiments, the compositions may be substantially free of oneor more, or all of non-branched chain amino acids. For example, thecompositions can be free of alanine, arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, glycine, histidine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,and/or tyrosine. In some embodiments, the compositions may besubstantially free of isoleucine and/or valine. The compositions can besubstantially free of any non-branched chain amino acids. The mass ormolar amount of a non-branched chain amino acid can be less than about0.01, 0.1, 0.5, 1, 2, 5, or 10% of the total composition or of the totalamino acids in the composition.

Pharmaceutically Active Agents

The subject compositions can further include one or morepharmaceutically active agents other than a methylxanthine PDEinhibitor. Examples of therapeutically active agents include ibuprofen,aldoril, and gemfebrozil, verapamil, maxzide, diclofenac and metrolol,maproltiline, triazolam and minoxidil. For example, the combinationcompositions can comprise a pharmaceutically active anti-diabetic agent,weight loss agent, or calcium regulation agent. U.S. Pat. No. 7,109,198and U.S. Patent Application No. 20090142336 describe a variety ofpharmaceutically active agents or therapeutically active agents suitablefor inclusion in a combination composition described herein. Examples ofanti-diabetic agents include biguanides (such as metformin),thiazoladinediones and meglitinides (such as repaglinide, pioglitazone,and rosiglitazone), alpha glucosidease inhibitors (such as acarbose),sulfonylureas (such as tolbutamide, acetohexamide, tolazamide,chlorpropamide, glipizide, glyburide, glimepiride, gliclazide),incretins, ergot alkaloids (such as bromocriptine), and DPP inhibitors(such as sitagliptin, vildagliptin, saxagliptin, lingliptin,dutogliptin, gemigliptin, alogliptin, and berberine). The anti-diabeticagent can be an oral anti-diabetic agent. The anti-diabetic agent canalso be injectable anti-diabetic drugs, including insulin, amylinanalogues (such as pramlintide), and inretin mimetics (such as exenatideand liraglutide). Examples of anti-obesity therapeutic agents includelipase inhibitors (such as Orlistat), dopaminergic, noradrenergic, andserotoninergic compounds, cannabinoid receptor antagonists (such asrimonabant), exenatide, pramlintide, and CNS agents (such astopimerate). These examples are provided for discussion purposes only,and are intended to demonstrate the broad scope of applicability of theinvention to a wide variety of drugs. It is not meant to limit the scopeof the invention in any way.

In some embodiments, a methylxanthine PDE inhibitor can be combined witha pair of pharmaceutically active agents as follow: glipizide andmetformin; glyburide and metformin; pioglitazone and glimepiride;pioglitazone and metformin; repaglinide and metformin; rosiglitazone andglimepiride; rosiglitazone and metformin; and sitagliptin and metformin.

The amount of pharmaceutical agent, or any other component used in acombination composition described herein, can be a used in an amountthat is sub-therapeutic. In some embodiments, using sub-therapeuticamounts of an agent or component can reduce the side-effects of theagent. Use of sub-therapeutic amounts can still be effective,particularly when used in synergy with other agents or components.

A sub-therapeutic amount of the agent or component can be such that itis an amount below which would be considered therapeutic. For example,FDA guidelines can suggest a specified level of dosing to treat aparticular condition, and a sub-therapeutic amount would be any levelthat is below the FDA suggested dosing level. The sub-therapeutic amountcan be about 1, 5, 10, 15, 20, 25, 30, 35, 50, 75, 90, or 95% less thanthe amount that is considered to be a therapeutic amount. Thetherapeutic amount can be assessed for individual subjects, or forgroups of subjects. The group of subjects can be all potential subjects,or subjects having a particular characteristic such as age, weight,race, gender, or physical activity level.

In the case of metformin hydrochloride, the physician suggested startingdose is 1000 mg daily, with subject specific dosing having a range of500 mg to a maximum of 2500 mg daily (metformin hydrochlorideextended-release tablets labelwww.accessdatafda.gov/drugsatfda_docs/label/2008/021574s010lbl.pdf). Theparticular dosing for a subject can be determined by a clinician bytitrating the dose and measuring the therapeutic response. Thetherapeutic dosing level can be determined by measuring fasting plasmaglucose levels and measuring glycosylated hemoglobin. A sub-therapeuticamount can be any level that would be below the recommended dosing ofmetformin. For example, if a subject's therapeutic dosing level isdetermined to be 700 mg daily, a dose of 600 mg would be asub-therapeutic amount. Alternatively, a sub-therapeutic amount can bedetermined relative to a group of subjects rather than an individualsubject. For example, if the average therapeutic amount of metformin forsubjects with weights over 300 lbs is 2000 mg, then a sub-therapeuticamount can be any amount below 2000 mg. In some embodiments, the dosingcan be recommended by a healthcare provider including, but not limitedto a patient's physician, nurse, nutritionist, pharmacist, or otherhealth care professional. A health care professional may include aperson or entity that is associated with the health care system.Examples of health care professionals may include surgeons, dentists,audiologists, speech pathologists, physicians (including generalpractitioners and specialists), physician assistants, nurses, midwives,pharmaconomists/pharmacists, dietitians, therapists, psychologists,physical therapists, phlebotomists, occupational therapists,optometrists, chiropractors, clinical officers, emergency medicaltechnicians, paramedics, medical laboratory technicians, radiographers,medical prosthetic technicians social workers, and a wide variety ofother human resources trained to provide some type of health careservice.

In the case of a methylxanthine, the therapeutically effective level ofthe methylxanthine can be a circulating level between about 44-111 μM,which corresponds to about 10-20 μg/mL. A sub-therapeutic level of themethylxanthine, such as theophylline or theobromine, can be anycirculating level below about 110, 100, 90, 80, 70, 60, 50, 44, 40, 35,30, 20, 10, 5, or 1 μM or 10 μg/mL. The sub-therapeutic level of themethylxanthine, such as theophylline or theobromine, in a subjectcomposition formulated for administration can be less than about 1, 5,10, 20, 30, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, or 250 mg ofthe methylxanthine.

Any of the components described herein, including leucine, HMB, KIC,theophylline, theobromine, and resveratrol may be used in a subjectcomposition in free form, purified from a natural source, and/orpurified or prepared from a synthetic source. The natural source can bean animal source or plant source. The components may be pure to at leastabout 95, 97, 99, 99.5, 99.9, 99.99, or 99.999%.

Dosing Amounts

The invention provides for compositions that are combinations ofisolated components, such as leucine, metabolites of leucine, such asHMB, methylxanthines, such as theophylline and theobromine, andresveratrol, that have been isolated from one or more sources. Theinvention provides for compositions that are enriched in leucine,metabolites of leucine, such as HMB, methylxanthines, such astheophylline and theobromine, and/or resveratrol. The components can beisolated from natural sources or created from synthetic sources and thenenriched to increase the purity of the components. For example,theophylline can be created from a synthetic source and then enriched byone or more purification methods. Additionally, leucine can be isolatedfrom a natural source and then enriched by one or more separations. Theisolated and enriched components, such as sildenafil and leucine, canthen be combined and formulated for administration to a subject.

In some embodiments, a composition comprises an amount of amethylxanthine or a PDE inhibitor (e.g., including but not limited totheophylline or theobromine). The amount of a methylxanthine may be asubtherapeutic amount, and/or an amount that is synergistic with one ormore other compounds in the composition or one or more of the compoundsadministered simultaneously or in close temporal proximity with thecomposition. In some embodiments, the methylxanthine or PDE inhibitor isadministered in a low dose, a medium dose, or a high dose, whichdescribes the relationship between two doses, and generally do notdefine any particular dose range. The compositions can be administeredto a subject such that the subject is administered a selected totaldaily dose of the composition. The total daily dose can be determined bythe sum of doses administered over a 24 hour period.

A dose, which may be a unit dose, can comprise about, more than about,or less than about 200, 250, 400, 500, 600, 700, 800, 900, 1000, 1100,1250, or more mg of leucine. The leucine may be free leucine. In someembodiments, a unit dose can comprise at least about 1000 mg of freeleucine. The composition may comprise between about 10-1250, 200-1250,or 500-1250 mg of leucine. A dose, which may be a unit dose, cancomprise about, more than about, or less than about 50, 100, 200, 250,400, 500, 600, 700, 800, 900, 1000 or more mg of a leucine metabolite,such as HMB or KIC. The leucine metabolite may be a free leucinemetabolite. The composition may comprise between about 10-900, 50-750,or 400-650 mg of the leucine metabolite, such as HMB or KIC. In someembodiments, a unit dose can comprise at least about 400 mg of free HMB.

In some embodiments, a daily dose of leucine can be about, less thanabout, or more than about 0.5-3.0 g/day (e.g. 0.5, 0.75, 1, 1.25, 1.5,1.75, 2, 2.5, 3, or more g/day). A daily dose of HMB can be about, lessthan about, or more than about 0.20-3.0 g/day (e.g. 0.2, 0.4, 0.5, 0.75,1, 1.5, 2, 2.5, 3, or more g/day). A daily dose of KIC can be about,less than about, or more than about 0.2-3.0 g/day (e.g. 0.2, 0.4, 0.5,0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, or more g/day).

A dose, which may be a unit dose, can comprise a methylxanthine PDEinhibitor, such as theophylline or theobromine, that can be about, morethan about, or less than about 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 20,40, 60, 80, 100, 200, 400, 800, 1000, or 1500 mg of the methylxanthinePDE inhibitor. The composition can comprise between about 1-100, 5-50,or 10-20 mg of the methylxanthine, such as theophylline or theobromine.In some embodiments, a unit dose can comprise at least about 20 mg oftheophylline or theobromine. In some embodiments, a unit dose cancomprise at least about 20 mg of theophylline.

In some embodiments, the composition comprises both theophylline andtheobromine, and the total amount of theophylline and theobromine can beabout, more than about, or less than about 0.01, 0.05, 0.1, 0.5, 1, 2,5, 10, 20, 40, 60, 80, 100, 200, 400, 800, 1000, or 1500 mg.

In other embodiments, a daily dose of a methylxanthine PDE inhibitor,such as theophylline or theobromine, can be about, more than about, orless than about 0.0001 mg/kg (mg of methylxanthine PDE inhibitor/kg ofthe subject receiving the dose), 0.005 mg/kg, 0.01 mg/kg, 0.5 mg/kg, 1mg/kg, 2.5 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 12.5 mg/kg, 15 mg/kg, 20mg/kg, 25 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, or more.

A dose, which may be a unit dose, can comprise about, less than about,or more than about 1, 5, 10, 25, 35, 50, 51, 75, 100, 150, 200, 250,300, 350, 400, 450, 500, or more mg of resveratrol. The composition maycomprise between about 5-500, 30-250, or 35-100 mg of resveratrol. Insome embodiments, a unit dose can comprise at least about 35 mg ofresveratrol.

A daily low dose of resveratrol may comprise about, less than about, ormore than about 0.5 mg/kg (mg of resveratrol/kg of the subject receivingthe dose), 1 mg/kg, 2.5 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 12.5 mg/kg,15 mg/kg, 20 mg/kg, 25 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, or more; adaily medium dose of resveratrol may comprise about, less than about, ormore than about 20 mg/kg, 25 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, 125mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, or more; and a dailyhigh dose of resveratrol may comprise about, less than about, or morethan about 150 mg/kg, 175 mg/kg, 200 mg/kg, 225 mg/kg, 250 mg/kg, 300mg/kg, 350 mg/kg, 400 mg/kg, or more.

In some embodiments, a composition, which may be formulated as a unitdose, can comprise (a) at least about 250 mg of leucine and/or at leastabout 100 mg of the one or more leucine metabolites and (b) comprises atleast about 5 mg of a methylxanthine, such as theophylline ortheobromine. The composition can further comprise at least about 35 mgof resveratrol.

In other embodiments, a composition, formulated as a unit dose, cancomprise (a) between about 250-1500 mg of leucine or 100-750 mg ofleucine metabolites and (b) 1-100 mg of a methylxanthine, such astheophylline or theobromine. In other embodiments, a composition,formulated as a unit dose, can comprise (a) between about 250-1500 mg ofleucine, 10-750 mg of leucine metabolites, and/or 50-750 mg of leucinemetabolites and (b) 1-100 mg of a methylxanthine, such as theophyllineor theobromine. In some embodiments, the methylxanthine is theophyllineand the composition comprises between about 5-50 mg of theophylline.

In some embodiments of the invention, the combination compositions canhave a specified ratio of leucine amino acids and/or metabolites thereofto a methylxanthine PDE inhibitor. The specified ratio can provide foreffective and/or synergistic treatment of pulmonary conditions, which,for example, may be measured as a reduction in NFκB protein expression,reduction in eotaxin secretion, reduction in IL1-β secretion, reductionin cellular IL6 content or secretion, reduction in adiponectin receptor1 protein expression, and reduction in adiponectin receptor 2 proteinexpression. The ratio of leucine amino acids and/or metabolites thereofto a selective PDE inhibitor activator can be a mass ratio, a molarratio, or a volume ratio.

In some embodiments, a composition can comprise (a) leucine and/ormetabolites thereof (including HMB) and (b) a methylxanthine (includingtheophylline and theobromine), where the mass ratio of (a) to (b) can beabout, less than about, or greater than about 0.1, 0.5, 1, 2, 5, 10, 15,25, 50, 75, 100, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,or 800. In some embodiments, the mass ratio of (a) to (b) is at leastabout 50. The composition can also comprise a minimal amount ofmethylxanthine, such as 5, 10 or 50 mg of the methylxanthine or a rangeof methylxanthine amount, such as 5-250 mg of methylxanthine.

In other embodiments, a composition can comprise (a) a methylxanthinePDE inhibitor (including theophylline and theobromine) and (b)resveratrol, where the mass ratio of (a) to (b) can be about, less thanabout, or greater than about 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 50, 100,200, 300, 350, 400, 450, 500, 550, 600, or 650.

In some embodiments, the composition can be formulated for inhalation.The composition formulated for inhalation can be formulated as a liquid.The composition formulated for inhalation can be housed within aninhaler or nebulizer. The inhaler or nebulizer can hold at least about10, 20, 40, 100, 500, 1000, or 2000 unit doses. A unit dose of a subjectcomposition can have a volume of about or at least about 0.1, 0.25, 0.5,1, or 5 mL. A unit dose of a subject composition can have a volume ofabout 0.5-5 mL, which may be administered in about 1-10 inhalations.

The composition formulated for inhalation can be formulated in a liquidform that comprises at least about, about, or less than about 0.25, 0.5,0.75, 1 mM or more of leucine.

The composition formulated for inhalation can be formulated in a liquidform that comprises at least about, about, or less than about 0.1, 0.25,0.5, 0.75, 1, 10, 20, 40, 60 μM or more of a leucine metabolite (such asHMB). The composition formulated for inhalation can be formulated in aliquid form that comprises at least about, about, or less than about0.25, 0.5, 0.75, 1 mM or more of KIC.

The composition formulated for inhalation can be formulated in a liquidform that comprises at least about, about, or less than about 0.1, 0.25,0.5, 0.75, 1, 10, 20, 40, 60, 80, 100, 120, 200, or 400 μM or more ofthe methylxanthine, such as theophylline or theobromine.

In some embodiments, the dosing of leucine, any metabolites of leucine,the PDE inhibitor (such as a methylxanthine), and resveratrol can bedesigned to achieve a specified physiological concentration orcirculating level of leucine, metabolites of leucine, a methylxanthineand/or resveratrol. The physiological concentration can be a circulatinglevel as measured in the blood stream of a subject. The subject can be ahuman or an animal. A selected dosing can be altered based on thecharacteristics of the subject, such as weight, rate of energymetabolism, genetics, ethnicity, height, or any other characteristic.

In some embodiments, a selected dose of a composition can beadministered to a subject such that the subject achieves a desiredcirculating level of the composition. The desired circulating level of acomponent may be either a therapeutically effective level or asub-therapeutic level.

The desired circulating level of the composition can be at least about0.25, 0.5, 0.75, 1 mM or more of leucine. The desired circulating levelof the composition can be at least about, less than about, or more thanabout 0.1, 0.25, 0.5, 0.75, 1, 10, 20, 40, 60 μM or more of a leucinemetabolite (such as HMB). The desired circulating level of thecomposition can be at least about 0.25, 0.5, 0.75, 1 mM or more of KIC.

The desired circulating level of the composition can be at least about,less than about, or more than about 0.1, 0.25, 0.5, 0.75, 1, 10, 20, 40,60, 80, 100, 120, 200, or 400 μM or more of the methylxanthine, such astheophylline or theobromine. The therapeutically effective level oftheophylline can be between 44-111 μM, which corresponds to about 10-20μg/mL. A sub-therapeutic level of theophylline can be any level belowabout 110, 100, 90, 80, 70, 60, 50, 44, 40, 35, 30, 20, 10, 5, or 1 μMor 10 μg/mL.

The desired circulating level of the composition can be at least about,less than about, or more than about 40, 60, 80, 100, 120, 150, 200, 300,400, 800, 1600, 3000, or 5000 nM or more of the resveratrol. Theselected dose can be chosen based on the characteristics of the subject,such as weight, height, ethnicity, or genetics.

In some embodiments, a composition comprises leucine and amethylxanthine in amounts that are effective to achieve a circulatinglevel of about 0.3-2 mM leucine and about 0.5-10 μM methylxanthine in asubject.

An oral dosing of about 1,125 mg leucine can achieve a circulating levelof leucine in a subject that is about 0.5 mM leucine. An oral dosing ofabout 300 mg leucine can achieve a circulating level of leucine in asubject that is about 0.25 mM.

An oral dosing of about 500 mg of HMB can achieve a circulating level ofHMB in a subject that is about 5 μM HMB. An oral dosing of about 100 mgof HMB can achieve a circulating level of HMB in a subject that is about0.8 μM HMB.

An oral dosing of about 1000 mg of theophylline can achieve acirculating level of theophylline in a subject that is about 110 μMtheophylline. An oral dosing of about 25-30 mg of theophylline canachieve a circulating level of theophylline in a subject that is about 1μM theophylline.

An oral dosing of about 1000 mg of theobromine can achieve a circulatinglevel of theobromine in a subject that is about 110 μM theobromine. Anoral dosing of about 25-30 mg of theobromine can achieve a circulatinglevel of theophylline in a subject that is about 1 μM theobromine.

An oral dosing of about 1100 mg of resveratrol can achieve a circulatinglevel of resveratrol in a subject that is about 0.5 mM resveratrol. Anoral dosing of about 50 mg of resveratrol can achieve a circulatinglevel of resveratrol in a subject that is about 200 nM resveratrol.

A dosing prepared for inhalation, which may be in liquid form, can beprepared at concentrations that are about 1, 1.5, 2, 2.5, or 3 timesgreater than the desired circulating concentration. For example, aformulation for inhalation may comprise about 0.5, 1, or 0.5-1 mMleucine, which can achieve a circulating level of leucine in thepulmonary tissue of a subject that is about 0.5 mM. A formulation forinhalation may comprise about 200, 400, or 200-400 nM resveratrol, whichcan achieve a circulating level of resveratrol in the pulmonary tissueof a subject that is about 200 nM. A formulation for inhalation maycomprise about 1, 2, or 1-2 μM methylxanthine, which can achieve acirculating level of methylxanthine in the pulmonary tissue of a subjectthat is about 1 μM.

In some embodiments, the compositions can be formulated to achieve adesired circulating molar or mass ratios achieved after administrationone or more compositions to a subject. The compositions can be acombination composition described herein. The molar ratio can beadjusted to account for the bioavailability, the uptake, and themetabolic processing of the one or more components of a combinationcomposition. For example, if the bioavailability of a component is low,then the molar amount of a that component can be increased relative toother components in the combination composition. In some embodiments,the circulating molar or mass ratio is achieved within about 0.1, 0.5,0.75, 1, 3, 5, or 10, 12, 24, or 48 hours after administration. Thecirculating molar or mass ratio can be maintained for a time period ofabout or greater than about 0.1, 1, 2, 5, 10, 12, 18, 24, 36, 48, 72, or96 hours.

In some embodiments, the circulating molar ratio of leucine to amethylxanthine is about, less than about, or greater than about 1, 5,10, 50, 100, 500, 1000, 5000, or 10000. In some embodiments, thecirculating molar ratio of HMB to a methylxanthine is about or greaterthan about, or less than about 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, or100. In some embodiments, the circulating molar ratio of amethylxanthine to resveratrol is about, less than about, or greater thanabout 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, or 100.

In some embodiments, a composition can comprise leucine and amethylxanthine in an amount that is effective in improving expressionlevel or secretion of one or more inflammatory or anti-inflammatorymarkers in a cell selected from the group consisting of (a) NFκB proteinexpression, (b) eotaxin, (c) IL1-β, and (d) cellular IL6 content orsecretion, (e) adiponectin receptor 1 protein expression, and (f)adiponectin receptor 2 protein expression. The composition can have anamount of methylxanthine that is less than the amount required toachieve a circulating level of about 40 μM. The composition can have anamount of methylxanthine that is effective to achieve a circulatinglevel of about 0.1-40 μM. The amount of methylxanthine can also begreater than at least about 5 mg.

Dosing Forms

The compositions described herein can be compounded into a variety ofdifferent dosage forms. It can be used orally as a tablet, chewabletablet, caplets, capsule, soft gelatin capsules, lozenges or solution.Alternatively, the compositions can be formulated for inhalation or forintravenous delivery. The compositions can also be formulated as a nasalspray or for injection when in solution form. In some embodiments, thecomposition may be a liquid composition suitable for oral consumption.

Compositions formulated for inhalation may be packaged in an inhalerusing techniques known in the art. An inhaler may be designed todispense 0.25, 0.5, or 1 unit dose per inhalation. An inhaler may have acanister that holds the subject composition formulated for inhalation, ametering valve that allows for a metered quantity of the formulation tobe dispensed with each actuation, and an actuator or mouthpiece thatallows for the device to be operated and direct the subject compositioninto the subject's lungs. The formulated composition may include aliquefied gas propellant and possibly stabilizing excipients. Theactuator may have a mating discharge nozzle that connects to thecanister and a dust cap to prevent contamination of the actuator. Uponactuation, the subject composition may be volatized, which results inthe formation of droplets of the subject composition. The droplets mayrapidly evaporate resulting in micrometer-sized particles that are theninhaled by the subject.

A protocol for treatment via inhalation can include preparation of anaerosol having particle sizes of predetermined mass medial aerodynamicdiameter (MMAD) between 3 and 8 μm delivered predominantly to theconducting and central lungs with or without overpressure using a jet,ultrasonic, electronic, vibrating porous plate, vibrating mesh nebulizeror energized dry powder inhaler. The jet or electronic nebulizers mayfurther be combined with airflow control and the aerosol may beadministered with overpressure. Nebulization devices and systems canallow for individualization of a delivered volumetric flow and vaporizedaerosol together with a controlled airflow and with airflow overpressureconditions into a treatment protocol suitable for treatment of a varietyof conditions including inflammatory pulmonary diseases.

The nebulizing system can comprise components, such as a jet orelectronic nebulizer, a compressor and an electronic control means thatcumulatively have properties that enable control of the breathingpattern by asserting a positive pressure (also called NIPPV) during theinhalation. This pressure can reduce the need for active breathing inCOPD patients, which results in much more effective and easier lungdelivery of the drugs combination to COPD patients having difficultybreathing or who are unable to breathe without oxygen.

Inhalers and methods for formulating compositions for inhalation aredescribed in are described in U.S. Pat. Nos. 5,069,204, 7,870,856 andU.S. Patent Application No. 2010/0324002, which are incorporated hereinby reference in its entirety.

Compositions of the invention suitable for oral administration can bepresented as discrete dosage forms, such as capsules, cachets, ortablets, or liquids or aerosol sprays each containing a predeterminedamount of an active ingredient as a powder or in granules, a solution,or a suspension in an aqueous or non-aqueous liquid, an oil-in-wateremulsion, or a water-in-oil liquid emulsion, including liquid dosageforms (e.g., a suspension or slurry), and oral solid dosage forms (e.g.,a tablet or bulk powder). Oral dosage forms may be formulated astablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilicsuspensions, liquids, gels, syrups, slurries, suspensions and the like,for oral ingestion by an individual or a patient to be treated. Suchdosage forms can be prepared by any of the methods of formulation. Forexample, the active ingredients can be brought into association with acarrier, which constitutes one or more necessary ingredients. Capsulessuitable for oral administration include push-fit capsules made ofgelatin, as well as soft, sealed capsules made of gelatin and aplasticizer, such as glycerol or sorbitol. The push-fit capsules cancontain the active ingredients in admixture with filler such as lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers. Optionally, the inventivecomposition for oral use can be obtained by mixing a composition a solidexcipient, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). In general,the compositions are prepared by uniformly and intimately admixing theactive ingredient with liquid carriers or finely divided solid carriersor both, and then, if necessary, shaping the product into the desiredpresentation. For example, a tablet can be prepared by compression ormolding, optionally with one or more accessory ingredients. Compressedtablets can be prepared by compressing in a suitable machine the activeingredient in a free-flowing form such as powder or granules, optionallymixed with an excipient such as, but not limited to, a binder, alubricant, an inert diluent, and/or a surface active or dispersingagent. Molded tablets can be made by molding in a suitable machine amixture of the powdered compound moistened with an inert liquid diluent.

The liquid forms, in which the formulations disclosed herein may beincorporated for administration orally or by injection, include aqueoussolution, suitably flavored syrups, aqueous or oil suspensions, andflavored emulsions with edible oils such as cottonseed oil, sesame oil,coconut oil, or peanut oil as well as elixirs and similar pharmaceuticalvehicles. Suitable dispersing or suspending agents for aqueoussuspensions include synthetic natural gums, such as tragacanth, acacia,alginate, dextran, sodium carboxymethyl cellulose, methylcellulose,polyvinylpyrrolidone or gelatin.

A subject can be treated by combination of an injectable composition andan orally ingested composition.

Liquid preparations for oral administration may take the form of, forexample, solutions, syrups or suspensions, or they may be presented as adry product for reconstitution with water or other suitable vehiclesbefore use. Such liquid preparations may be prepared by conventionalmeans with pharmaceutically acceptable additives such as suspendingagents (e.g., sorbitol syrup, methyl cellulose or hydrogenated ediblefats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters or ethyl alcohol); preservatives(e.g., methyl or propyl p-hydroxybenzoates or sorbic acid); andartificial or natural colors and/or sweeteners.

The preparation of pharmaceutical compositions of this invention,including oral and inhaled formulations, can be conducted in accordancewith generally accepted procedures for the preparation of pharmaceuticalpreparations. See, for example, Remington's Pharmaceutical Sciences 18thEdition (1990), E. W. Martin ed., Mack Publishing Co., PA. Depending onthe intended use and mode of administration, it may be desirable toprocess the magnesium-counter ion compound further in the preparation ofpharmaceutical compositions. Appropriate processing may include mixingwith appropriate non-toxic and non-interfering components, sterilizing,dividing into dose units, and enclosing in a delivery device.

This invention further encompasses anhydrous compositions and dosageforms comprising an active ingredient, since water can facilitate thedegradation of some compounds. For example, water may be added (e.g.,5%) in the arts as a means of simulating long-term storage in order todetermine characteristics such as shelf-life or the stability offormulations over time. Anhydrous compositions and dosage forms of theinvention can be prepared using anhydrous or low moisture containingingredients and low moisture or low humidity conditions. Compositionsand dosage forms of the invention which contain lactose can be madeanhydrous if substantial contact with moisture and/or humidity duringmanufacturing, packaging, and/or storage is expected. An anhydrouscomposition may be prepared and stored such that its anhydrous nature ismaintained. Accordingly, anhydrous compositions may be packaged usingmaterials known to prevent exposure to water such that they can beincluded in suitable formulary kits. Examples of suitable packaginginclude, but are not limited to, hermetically sealed foils, plastic orthe like, unit dose containers, blister packs, and strip packs.

An ingredient described herein can be combined in an intimate admixturewith a pharmaceutical carrier according to conventional pharmaceuticalcompounding techniques. The carrier can take a wide variety of formsdepending on the form of preparation desired for administration. Inpreparing the compositions for an oral dosage form, any of the usualpharmaceutical media can be employed as carriers, such as, for example,water, glycols, oils, alcohols, flavoring agents, preservatives,coloring agents, and the like in the case of oral liquid preparations(such as suspensions, solutions, and elixirs) or aerosols; or carrierssuch as starches, sugars, micro-crystalline cellulose, diluents,granulating agents, lubricants, binders, and disintegrating agents canbe used in the case of oral solid preparations, in some embodimentswithout employing the use of lactose. For example, suitable carriersinclude powders, capsules, and tablets, with the solid oralpreparations. If desired, tablets can be coated by standard aqueous ornonaqueous techniques.

Some examples of materials which may serve as pharmaceuticallyacceptable carriers include: (1) sugars, such as lactose, glucose andsucrose; (2) starches, such as corn starch and potato starch; (3)cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10)glycols, such as propylene glycol; (11) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyloleate and ethyl laurate; (13) agar; (14) buffering agents, such asmagnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19)ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxiccompatible substances employed in pharmaceutical formulations.

Binders suitable for use in dosage forms include, but are not limitedto, corn starch, potato starch, or other starches, gelatin, natural andsynthetic gums such as acacia, sodium alginate, alginic acid, otheralginates, powdered tragacanth, guar gum, cellulose and its derivatives(e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulosecalcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methylcellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose,microcrystalline cellulose, and mixtures thereof.

Lubricants which can be used to form compositions and dosage forms ofthe invention include, but are not limited to, calcium stearate,magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol,mannitol, polyethylene glycol, other glycols, stearic acid, sodiumlauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil,cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethylaureate, agar, ormixtures thereof. Additional lubricants include, for example, a syloidsilica gel, a coagulated aerosol of synthetic silica, or mixturesthereof. A lubricant can optionally be added, in an amount of less thanabout 1 weight percent of the composition.

Lubricants can be also be used in conjunction with tissue barriers whichinclude, but are not limited to, polysaccharides, polyglycans,seprafilm, interceed and hyaluronic acid.

Disintegrants may be used in the compositions of the invention toprovide tablets that disintegrate when exposed to an aqueousenvironment. Too much of a disintegrant may produce tablets which maydisintegrate in the bottle. Too little may be insufficient fordisintegration to occur and may thus alter the rate and extent ofrelease of the active ingredient(s) from the dosage form. Thus, asufficient amount of disintegrant that is neither too little nor toomuch to detrimentally alter the release of the active ingredient(s) maybe used to form the dosage forms of the compounds disclosed herein. Theamount of disintegrant used may vary based upon the type of formulationand mode of administration, and may be readily discernible to those ofordinary skill in the art. About 0.5 to about 15 weight percent ofdisintegrant, or about 1 to about 5 weight percent of disintegrant, maybe used in the pharmaceutical composition. Disintegrants that can beused to form compositions and dosage forms of the invention include, butare not limited to, agar-agar, alginic acid, calcium carbonate,microcrystalline cellulose, croscarmellose sodium, crospovidone,polacrilin potassium, sodium starch glycolate, potato or tapioca starch,other starches, pre-gelatinized starch, other starches, clays, otheralgins, other celluloses, gums or mixtures thereof.

Examples of suitable fillers for use in the compositions and dosageforms disclosed herein include, but are not limited to, talc, calciumcarbonate (e.g., granules or powder), microcrystalline cellulose,powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol,starch, pre-gelatinized starch, and mixtures thereof.

When aqueous suspensions and/or elixirs are desired for oraladministration, the active ingredient therein may be combined withvarious sweetening or flavoring agents, coloring matter or dyes and, ifso desired, emulsifying and/or suspending agents, together with suchdiluents as water, ethanol, propylene glycol, glycerin and variouscombinations thereof.

The tablets can be uncoated or coated by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearate canbe employed. Formulations for oral use can also be presented as hardgelatin capsules wherein the active ingredient is mixed with an inertsolid diluent, for example, calcium carbonate, calcium phosphate orkaolin, or as soft gelatin capsules wherein the active ingredient ismixed with water or an oil medium, for example, peanut oil, liquidparaffin or olive oil.

In one embodiment, the composition may include a solubilizer to ensuregood solubilization and/or dissolution of the compound of the presentinvention and to minimize precipitation of the compound of the presentinvention. This can be especially important for compositions fornon-oral use, e.g., compositions for injection. A solubilizer may alsobe added to increase the solubility of the hydrophilic drug and/or othercomponents, such as surfactants, or to maintain the composition as astable or homogeneous solution or dispersion.

The composition can further include one or more pharmaceuticallyacceptable additives and excipients. Such additives and excipientsinclude, without limitation, detackifiers, anti-foaming agents,buffering agents, polymers, antioxidants, preservatives, chelatingagents, viscomodulators, tonicifiers, flavorants, colorants, odorants,opacifiers, suspending agents, binders, fillers, plasticizers,lubricants, and mixtures thereof. A non-exhaustive list of examples ofexcipients includes monoglycerides, magnesium stearate, modified foodstarch, gelatin, microcrystalline cellulose, glycerin, stearic acid,silica, yellow beeswax, lecithin, hydroxypropylcellulose, croscarmellosesodium, and crospovidone.

The compositions described herein can also be formulated asextended-release, sustained-release or time-release such that one ormore components are released over time. Delayed release can be achievedby formulating the one or more components in a matrix of a variety ofmaterials or by microencapsulation. The compositions can be formulatedto release one or more components over a time period of 1, 4, 6, 8, 12,16, 20, 24, 36, or 48 hours. The release of the one or more componentscan be at a constant or changing rate.

In some embodiments, a subject composition described herein can beformulated in as matrix pellets in which particles of the subjectcomposition are embedded in a matrix of water-insoluble plastic andwhich are enclosed by a membrane of water-insoluble plastic containingembedded particles of lactose, produces and maintains plasma levels ofthe subject composition within the targeted therapeutic range. In otherembodiments, a subject composition can be formulated as a sustainedrelease tablet obtained by coating core granules composed mainly of thesubject composition with a layer of a coating film composed of ahydrophobic material and a plastic excipient and optionally containingan enteric polymer material to form coated granules and then bycompressing the coated granules together with a disintegratingexcipient. Sustained release formulations are described in U.S. Pat.Nos. 4,803,080, and 6,426,091, which are herein incorporated byreference in its entirety.

Using the controlled release dosage forms provided herein, the one ormore cofactors can be released in its dosage form at a slower rate thanobserved for an immediate release formulation of the same quantity ofcomponents. In some embodiments, the rate of change in the biologicalsample measured as the change in concentration over a defined timeperiod from administration to maximum concentration for an controlledrelease formulation is less than about 80%, 70%, 60%, 50%, 40%, 30%,20%, or 10% of the rate of the immediate release formulation.Furthermore, in some embodiments, the rate of change in concentrationover time is less than about 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%of the rate for the immediate release formulation.

In some embodiments, the rate of change of concentration over time isreduced by increasing the time to maximum concentration in a relativelyproportional manner. For example, a two-fold increase in the time tomaximum concentration may reduce the rate of change in concentration byapproximately a factor of 2. As a result, the one or more cofactors maybe provided so that it reaches its maximum concentration at a rate thatis significantly reduced over an immediate release dosage form. Thecompositions of the present invention may be formulated to provide ashift in maximum concentration by 24 hours, 16 hours, 8 hours, 4 hours,2 hours, or at least 1 hour. The associated reduction in rate of changein concentration may be by a factor of about 0.05, 0.10, 0.25, 0.5 or atleast 0.8. In certain embodiments, this is accomplished by releasingless than about 30%, 50%, 75%, 90%, or 95% of the one or more cofactorsinto the circulation within one hour of such administration.

Optionally, the controlled release formulations exhibit plasmaconcentration curves having initial (e.g., from 2 hours afteradministration to 4 hours after administration) slopes less than 75%,50%, 40%, 30%, 20% or 10% of those for an immediate release formulationof the same dosage of the same cofactor.

In some embodiments, the rate of release of the cofactor as measured indissolution studies is less than about 80%, 70%, 60% 50%, 40%, 30%, 20%,or 10% of the rate for an immediate release formulation of the samecofactor over the first 1, 2, 4, 6, 8, 10, or 12 hours.

The controlled release formulations provided herein can adopt a varietyof formats. In some embodiments, the formulation is in an oral dosageform, including liquid dosage forms (e.g., a suspension or slurry), andoral solid dosage forms (e.g., a tablet or bulk powder), such as, butnot limited to those, those described herein.

The controlled release tablet of a formulation disclosed herein can beof a matrix, reservoir or osmotic system. Although any of the threesystems is suitable, the latter two systems can have more optimalcapacity for encapsulating a relatively large mass, such as for theinclusion of a large amount of a single cofactor, or for inclusion of aplurality of cofactors, depending on the genetic makeup of theindividual. In some embodiments, the slow-release tablet is based on areservoir system, wherein the core containing the one or more cofactorsis encapsulated by a porous membrane coating which, upon hydration,permits the one or more cofactors to diffuse through. Because thecombined mass of the effective ingredients is generally in gramquantity, an efficient delivery system can provide optimal results.

Thus, tablets or pills can also be coated or otherwise compounded toprovide a dosage form affording the advantage of prolonged action. Forexample, the tablet or pill can comprise an inner dosage an outer dosagecomponent, the latter being in the form of an envelope over the former.The two components can be separated by an enteric layer which serves toresist disintegration in the stomach and permits the inner component topass intact into the duodenum or to be delayed in release. A variety ofmaterials can be used for such enteric layers or coatings such materialsincluding a number of polymeric acids and mixtures of polymeric acidswith such materials as shellac, cetyl alcohol and cellulose acetate. Insome embodiments, a formulation comprising a plurality of cofactors mayhave different cofactors released at different rates or at differenttimes. For example, there can be additional layers of cofactorsinterspersed with enteric layers.

Methods of making sustained release tablets are known in the art, e.g.,see U.S. Patent Publications 2006/051416 and 2007/0065512, or otherreferences disclosed herein. Methods such as described in U.S. Pat. Nos.4,606,909, 4,769,027, 4,897,268, and 5,395,626 can be used to preparesustained release formulations of the one or more cofactors determinedby the genetic makeup of an individual. In some embodiments, theformulation is prepared using OROS® technology, such as described inU.S. Pat. Nos. 6,919,373, 6,923,800, 6,929,803, and 6,939,556. Othermethods, such as described in U.S. Pat. Nos. 6,797,283, 6,764,697, and6,635,268, can also be used to prepare the formulations disclosedherein.

In some embodiments, the compositions can be formulated in a foodcomposition. For example, the compositions can be a beverage or otherliquids, solid food, semi-solid food, with or without a food carrier.For example, the compositions can include a black tea supplemented withany of the compositions described herein. The composition can be a dairyproduct supplemented any of the compositions described herein. In someembodiments, the compositions can be formulated in a food composition.For example, the compositions can comprise a beverage, solid food,semi-solid food, or a food carrier.

In some embodiments, liquid food carriers, such as in the form ofbeverages, such as supplemented juices, coffees, teas, sodas, flavoredwaters, and the like can be used. For example, the beverage can comprisethe formulation as well as a liquid component, such as various deodorantor natural carbohydrates present in conventional beverages. Examples ofnatural carbohydrates include, but are not limited to, monosaccharidessuch as, glucose and fructose; disaccharides such as maltose andsucrose; conventional sugars, such as dextrin and cyclodextrin; andsugar alcohols, such as xylitol and erythritol. Natural deodorant suchas taumatin, stevia extract, levaudioside A, glycyrrhizin, and syntheticdeodorant such as saccharin and aspartame may also be used. Agents suchas flavoring agents, coloring agents, and others can also be used. Forexample, pectic acid and the salt thereof, alginic acid and the saltthereof, organic acid, protective colloidal adhesive, pH controllingagent, stabilizer, a preservative, glycerin, alcohol, or carbonizingagents can also be used. Fruit and vegetables can also be used inpreparing foods or beverages comprising the formulations discussedherein.

Alternatively, the compositions can be a snack bar supplemented with anyof the compositions described herein. For example, the snack bar can bea chocolate bar, a granola bar, or a trail mix bar. In yet anotherembodiment, the present dietary supplement or food compositions areformulated to have suitable and desirable taste, texture, and viscosityfor consumption. Any suitable food carrier can be used in the presentfood compositions. Food carriers of the present invention includepractically any food product. Examples of such food carriers include,but are not limited to food bars (granola bars, protein bars, candybars, etc.), cereal products (oatmeal, breakfast cereals, granola,etc.), bakery products (bread, donuts, crackers, bagels, pastries,cakes, etc.), beverages (milk-based beverage, sports drinks, fruitjuices, alcoholic beverages, bottled waters), pastas, grains (rice,corn, oats, rye, wheat, flour, etc.), egg products, snacks (candy,chips, gum, chocolate, etc.), meats, fruits, and vegetables. In anembodiment, food carriers employed herein can mask the undesirable taste(e.g., bitterness). Where desired, the food composition presented hereinexhibit more desirable textures and aromas than that of any of thecomponents described herein. For example, liquid food carriers may beused according to the invention to obtain the present food compositionsin the form of beverages, such as supplemented juices, coffees, teas,and the like. In other embodiments, solid food carriers may be usedaccording to the invention to obtain the present food compositions inthe form of meal replacements, such as supplemented snack bars, pasta,breads, and the like. In yet other embodiments, semi-solid food carriersmay be used according to the invention to obtain the present foodcompositions in the form of gums, chewy candies or snacks, and the like.

The dosing of the combination compositions can be administered about,less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore times a daily. A subject can receive dosing for a period of about,less than about, or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14 or more days, weeks or months. A unit dose can be afraction of the daily dose, such as the daily dose divided by the numberof unit doses to be administered per day. A unit dose can be a fractionof the daily dose that is the daily dose divided by the number of unitdoses to be administered per day and further divided by the number ofunit doses (e.g. tablets) per administration. The number of unit dosesper administration may be about, less than about, or more than about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The number of doses per day may beabout, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more. The number of unit doses per day may be determined bydividing the daily dose by the unit dose, and may be about, less thanabout, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 6, 17, 18, 19, 20, or more unit doses per day. For example, a unitdose can be about ½, ⅓, ¼, ⅕, ⅙, 1/7, ⅛, 1/9, 1/10. A unit dose can beabout one-third of the daily amount and administered to the subjectthree times daily. A unit dose can be about one-half of the daily amountand administered to the subject twice daily. A unit dose can be aboutone-fourth of the daily amount with two unit doses administered to thesubject twice daily. In some embodiments, a unit dose comprises about,less than about, or more than about 50 mg resveratrol. In someembodiments, a unit dose comprises about, less than about, or more thanabout 550 mg leucine. In some embodiments, a unit dose comprises about,less than about, or more than about 200 mg of one or more leucinemetabolites.

In some embodiments, a unit dose (e.g. a unit dose comprising one ormore leucine metabolites, such as HMB) is administered as one unit dosetwo timer per day. A unit dose may comprise more than one capsule,tablet, vial, or entity.

Compositions disclosed herein can further comprise a flavorant and canbe a solid, liquid, gel or emulsion.

Methods

The subject composition is particularly useful for amelioratinginflammatory responses elicited during onset of a pulmonary condition.In one embodiment, the invention provides for methods for reducingexpression level or secretion of an inflammatory marker including butnot limited to NFκB, eotaxin, IL1-β, and IL6, or increasing expressionlevel or secretion of an anti-inflammatory marker including but notlimited to adiponectin receptor 1 and adiponectin receptor 2, comprisingcontacting a lung endothelial cell with any of the subject compositions.

In various embodiments of the invention, a composition is administeredto the subject in an amount that delivers synergizing amounts of leucineand/or a metabolite thereof, a methylxanthine PDE inhibitor, and/orresveratrol sufficient treat pulmonary conditions of the subject.

In some embodiments, the amounts of the methylxanthine, such astheophylline or theobromine, in the composition, if administered to asubject alone and without leucine, a leucine metabolite, or resveratrol,would cause no therapeutic effect in the subject. Additionally, theamounts of leucine, a leucine metabolite, or resveratrol, ifadministered to the subject without the methylxanthine, would have notherapeutic effect on the subject. However, when the methylxanthine isadministered in conjunction with either leucine, a leucine metabolite,or resveratrol, a therapeutic effect is observed.

Accordingly, the invention provides a method for administering acomposition comprising (a) leucine and/or one or more metabolitesthereof and (b) a methylxanthine present in a sub-therapeutic amount,wherein the composition is effective in increasing treating pulmonaryconditions by at least about 5 fold as compared to that of component (b)when it is used alone. The amount of leucine in the composition may alsobe a sub-therapeutic amount.

The invention also provides a method for administering a compositioncomprising (a) leucine and/or one or more metabolites thereof, (b) amethylxanthine present in a sub-therapeutic amount, and (c) resveratrolwherein the composition is effective in increasing treating pulmonaryconditions by at least about 5 fold as compared to that of component (b)when it is being used alone.

Quantification of the therapeutic effect can show that the effect of acomposition that comprises (a) a methylxanthine and (b) leucine or aleucine metabolite is greater than the predicted effect of administering(a) or (b) alone, assuming simple additive effects of (a) and (b), andthus the effect is synergistic. The synergistic effect can be quantifiedas the measured effect above the predicted simple additive effect of thecomponents of the composition. For example, if administration ofcomponent (a) alone yields an effect of 10% relative to control,administration of component (b) alone yields an effect of 15% relativeto control, and administration of a composition comprising both (a) and(b) yields an effect of 60% relative to control, the synergistic effectwould be 60%-(15%+10%), or 35%.

At an in vitro level, the beneficial effects of the compositions can bemeasured on cultured lung endothelial cells, such as mouse primary lungendothelial cells, treated with corresponding concentrations of thecompositions described herein. Analysis of the cells can allow forquantification of the beneficial effects. For example, western blots orELISA assays can be performed to measure the quantity of (a) reductionin NFκB protein expression, (b) eotaxin secretion, (c) reduction inIL1-β secretion, (d) reduction in cellular IL6 content or secretion, (e)increase in adiponectin receptor 1 protein expression, and/or (f)increase in adiponectin receptor 2 protein expression in the cell as aresult of treatment with a composition described herein.

Accordingly, the multi-component compositions described herein (such astheophylline/leucine, theophylline/leucine/resveratrol,theobromine/leucine, and theobromine/leucine/resveratrol) may have asynergistic effect on (a) reduction in NFκB protein expression, (b)eotaxin secretion, (c) reduction in IL1-β secretion, (d) reduction incellular IL6 content or secretion, (e) increase in adiponectin receptor1 protein expression, and/or (f) increase in adiponectin receptor 2protein expression that is at least about 10, 20, 50, 100, 200, or 300%.

The output of the pathways and beneficial effects achieved in a subjectcan be measured using one or more methods, disclosed herein and/or knownin the art. For example, measurements of COPD and asthma may be madeusing spirometry, refinements of spirometry, such as measurement of FEV6and inspiratory capacity; measurements of functional outcomes, such asdyspnea indexes and exercise tests; and measurements of global-clinicaloutcomes, such as quality of life questionnaires and assessment offrequency and severity of acute exacerbations. In some embodiments,administration of the compositions described herein to a subject caneffect spirometry values, lung capacity, and functional outcomemeasurements in the subject that are improvements of at least about 10,30, 50, 100, 200 or 300% greater relative to no administration of thecompositions or prior to administration of the compositions. The effectsmay also be synergistic effects of at least about 10, 20, 50, 100, 200,or 300%.

The compositions can be administered to a subject orally or by any othermethods. Methods of oral administration include administering thecomposition as a liquid, a solid, or a semi-solid that can be taken inthe form of a dietary supplement or a food stuff.

The compositions can be administered periodically. For example, thecompositions can be administered one, two, three, four times a day, oreven more frequent. The subject can be administered every 1, 2, 3, 4, 5,6 or 7 days. In some embodiments, the compositions are administeredthree times daily. The administration can be concurrent with meal timeof a subject. The period of treatment or diet supplementation can be forabout 1, 2, 3, 4, 5, 6, 7, 8, or 9 days, 2 weeks, 1-11 months, or 1year, 2 years, 5 years or even longer. In some embodiments of theinvention, the dosages that are administered to a subject can change orremain constant over the period of treatment. For example, the dailydosing amounts can increase or decrease over the period ofadministration.

The length of the period of administration and/or the dosing amounts canbe determined by a physician or any other type of clinician. Thephysician or clinician can observe the subject's response to theadministered compositions and adjust the dosing based on the subject'sperformance. For example, dosing for subjects that show reduced effectsin energy regulation can be increased to achieve desired results.

In some embodiments, the compositions administered to a subject can beoptimized for a given subject. For example, the ratio of branched chainamino acids to a sirtuin pathway activator or the particular componentsin a combination composition can be adjusted. The ratio and/orparticular components can be selected after evaluation of the subjectafter being administered one or more compositions with varying ratios ofbranched chain amino acids to a sirtuin pathway activator or varyingcombination composition components.

Another aspect of the invention provides for achieving desired effectsin one or more subjects after administration of a combinationcomposition described herein for a specified time period. For example,the beneficial effects of the compositions described herein can beobserved after administration of the compositions to the subject for 1,2, 3, 4, 6, 8, 10, 12, 24, or 52 weeks.

The invention provides for a method of treating subjects, comprisingidentifying a pool of subjects amenable to treatment. The identifyingstep can include one or more screening tests or assays. For example,subjects that are identified as asthmatic, diabetic or that have aboveaverage or significantly greater than average body mass indices and/orweight can be selected for treatment. The identifying step can include agenetic test that identifies one or more genetic variants that suggestthat the subject is amenable to treatment. The identified subjects canthen be treated with one or more compositions described herein. Forexample, they may be treated with a combination composition comprising asirtuin pathway activator and a branched-chain amino acid.

The invention also provides for methods of manufacturing thecompositions described herein. In some embodiments, the manufacture of acomposition described herein comprises mixing or combining two or morecomponents. These components can include a PDE inhibitor or sirtuin orAMPK pathway activator (such as a polyphenol or polyphenol precursorlike resveratrol, or a methylxanthine), and leucine or metabolitesthereof (such as HMB, or KIC). The amount or ratio of components can bethat as described herein. For example, the mass ratio of leucinecompared with resveratrol can be greater than about 80.

In some embodiments, the compositions can be combined or mixed with apharmaceutically active agent, a carrier, and/or an excipient. Examplesof such components are described herein. The combined compositions canbe formed into a unit dosage as tablets, capsules, gel capsules,slow-release tablets, or the like.

In some embodiments, the composition is prepared such that a solidcomposition containing a substantially homogeneous mixture of the one ormore components is achieved, such that the one or more components aredispersed evenly throughout the composition so that the composition maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules.

Kits

The invention also provides kits. The kits include one or morecompositions described herein, in suitable packaging, and may furthercomprise written material that can include instructions for use,discussion of clinical studies, listing of side effects, and the like.Such kits may also include information, such as scientific literaturereferences, package insert materials, clinical trial results, and/orsummaries of these and the like, which indicate or establish theactivities and/or advantages of the composition, and/or which describedosing, administration, side effects, drug interactions, or otherinformation useful to the health care provider. Such information may bebased on the results of various studies, for example, studies usingexperimental animals involving in vivo models and studies based on humanclinical trials. A kit may comprise one or more unit doses describedherein. In some embodiments, a kit comprises about, less than about, ormore than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 30, 31, 60, 90, 120, 150, 180, 210, or more unit doses.Instructions for use can comprise dosing instructions, such asinstructions to take 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more unit doses1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times per day. For example, a kitmay comprise a unit dose supplied as a tablet, with each tablet packageseparately, multiples of tablets packaged separately according to thenumber of unit doses per administration (e.g. pairs of tablets), or alltablets packaged together (e.g. in a bottle). As a further example, akit may comprise a unit dose supplied as a bottled drink, the kitcomprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 24, 28, 36,48, 72, or more bottles.

The kit may further contain another agent. In some embodiments, thecompound of the present invention and the agent are provided as separatecompositions in separate containers within the kit. In some embodiments,the compound of the present invention and the agent are provided as asingle composition within a container in the kit. Suitable packaging andadditional articles for use (e.g., measuring cup for liquidpreparations, foil wrapping to minimize exposure to air, and the like)are known in the art and may be included in the kit. Kits describedherein can be provided, marketed and/or promoted to health providers,including physicians, nurses, pharmacists, formulary officials, and thelike. Kits may also, in some embodiments, be marketed directly to theconsumer.

EXAMPLES Example 1—Effects of Leucine, Theophylline, Theobromine andResveratrol on Indicators of Pulmonary Conditions

Techniques

Cell Culture:

Mouse primary lung endothelial cells (MLEC) were obtained from CellBiologics (Chicago, Ill.). Cells were grown to confluence in T75 cultureflasks precoated with 0.2% gelatin. Cells were grown in Dulbecco'smodified eagle's medium (DMEM) containing a growth factor supplement(Cell Biologics #M1 166) which included 5% fetal bovine serum (FBS),heparin, EGF, hydrocortisone, L-glutamine and antibiotics at 37° C. in5% CO₂.

Adipocyte Conditioned Media Experiments:

3T3L1 adipocytes (passage 11 to 13) were grown and differentiated on6-well plates and treated for 48 h with the treatments indicated in“Results”. Media was then collected and pooled together for eachtreatment group. Pooled media was then used to treat confluent MLEC for24 h in lieu of direct treatments. The MLEC media from each cellreplicate was collected, aliquoted and stored in −20° C. for furtherexperiments. The cells (MLEC and adipocytes) were washed once withice-cold HBSS, then ice-cold RIPA buffer plus Protease and PhosphataseInhibitors (Sigma) was added. The cell extract was then incubated on icefor 10 min, scraped, transferred to a new microcentrifuge tube,homogenized for 5 sec and then centrifuged for 10 min at 12,000×g at 4°C. Then the clear supernatant from each cell replicate was aliquoted andstored at −20° C. for further experiments, as indicated below.

Western Blot:

NFκB and phospho-NFκB antibodies were obtained from Cell Signaling(Danvers, Mass.). Cells were treated as indicated in results and thecellular fractions were prepared using standard methods. Protein wasmeasured by BCA kit (Thermo Scientific). For Western blot, 2 μg (forNFκB) or 4 μg (for phospho-NFκB) of protein from the cell lysate wasresolved on 10% Tris/HCL polyacrylamide gels (Criterion precast gel,Bio-Rad Laboratories, Hercules, Calif.), transferred to PVDF membranes(NFκB and phospho-NFκB), incubated in blocking buffer (3% BSA in TBS)and then incubated with primary antibody, washed and incubated withsecondary horseradish peroxidase-conjugated antibody. Visualization andchemiluminescent detection was conducted using BioRad ChemiDocinstrumentation and software (Bio-Rad Laboratories, Hercules, Calif.)and band intensity was assessed using Image Lab 4.0 (Bio-RadLaboratories, Hercules, Calif.), with correction for background andloading controls. NFκB was detected at 60 kDA, and phospho-NFκB at 52-59kDA.

Cytokines:

Interleukin 1-β, eotaxin, and interleukin 6 were all measured viaenzyme-linked immunosorbent assay using specific antibodies for eachcytokine (Abcam, Cambridge, Mass.) and a horseradish peroxidase-basedsecond antibody detection system, with chromogenic detection at 450 nmusing a microplate reader (Synergy HT, BioTek Instruments, Winooski,Vt.).

Results

Mouse primary lung endothelial cells were treated with combinations oftheophylline (1 μM), theobromine (1 μM), leucine (0.5 mM), andresveratrol (200 nM). Each of theophylline, theobromine, leucine, andresveratrol may be purchased from appropriate vendors in the formindicated or salts of the forms indicated. Following treatment, westernblots or ELISA assays were performed on the MLEC to determine the levelof various indicators of airway diseases, such as NFκB proteinexpression, eotaxin secretion, IL1-β secretion, cellular IL6 content orsecretion, adiponectin receptor 1 protein expression, and adiponectinreceptor 2 protein expression.

Treatment with 0.5 mM leucine corresponds to a circulating level of thesame molarity achieved by orally administering about 1,125 mg of leucineto a human subject. Treatment with 0.25 mM leucine corresponds to acirculating level of the same molarity achieved by orally administeringabout 300 mg of leucine to a human subject.

Treatment with 110 μM theophylline corresponds to a circulating level ofthe same molarity achieved by orally administering about 1000 mg oftheophylline to a human subject. Treatment with 1 μM theophyllinecorresponds to a circulating level of the same molarity achieved byorally administering about 25-30 mg of theophylline to a human subject.

Treatment with 0.5 mM resveratrol corresponds to a circulating level ofthe same molarity achieved by orally administering about 1100 mg ofresveratrol to a human subject. Treatment with 200 nM resveratrolcorresponds to a circulating level of the same molarity achieved byorally administering about 50 mg of resveratrol to a human subject.

Theophylline exerted no independent effect on NFκB protein expressionrelative to control (FIG. 1), but the theophylline/leucine andtheophylline/leucine/resveratrol combinations resulted in significantreductions in NFκB (p<0.02); there was no significant difference betweentheophylline/leucine and theophylline/leucine/resveratrol treatments.Treatment of MLEC with a combination of resveratrol and leucine had noeffect on the NFκB levels relative to control. Phospho-NFκB exhibitedsimilar trends. As shown by this data, the combination of theophyllinewith leucine and theophylline with leucine and resveratrol has asynergistic effect because treatment with alone with (a) theophylline or(b) leucine and resveratrol had no effect on NFκB or phospho-NFκBlevels. The synergistic effect of combining theophylline with leucine orleucine and resveratrol was an improvement of at least about 22%relative to baseline (treatment with theophylline alone or leucine andresveratrol alone).

These effects on NFκB and phospho-NFκB were not recapitulated in theconditioned media experiments (FIG. 2 and FIG. 3), indicating that theseare direct effects on MLEC and not mediated by alterations in cytokinesecretion by adipocytes. The conditioned media experiments utilizedmedia prepared from adipocytes treated with a control, theophylline,theophylline/leucine, resveratrol/leucine, andtheophylline/resveratrol/leucine at the concentrations indicated above.

In contrast, although this low concentration of theophylline exerted nosignificant independent effect on any of the inflammatory mediatorsstudied, the leucine/theophylline and leucine/theophylline/resveratrolcombinations resulted in marked reductions (˜70%), relative to control,in IL1-β secretion (p<0.001; FIG. 4), with a smaller reduction foundwith the leucine/resveratrol combination in the absence of theophylline(p<0.01). TNFα used as a positive control to stimulate secretion of thecytokine, and an increase in IL1-β secretion was observed when MLEC wastreated with TNFα (p<0.01).

As can be seen in FIG. 4, the combined treatment of theophylline with(a) leucine or (b) leucine and resveratrol yielded a synergistic effecton IL1-β secretion. Treatment with theophylline alone had no effectrelative to the control and treatment with leucine/resveratrol had aneffect of about 20% relative to control. In comparison, the combinedtreatment of theophylline with (a) leucine or (b) leucine andresveratrol had an improvement of about 70% relative to baseline. Inthis case, the synergistic effect is at least about 50% relative to thecontrol.

Similarly, the leucine/theophylline combination elicited a significant44% decrease in the secretion of the chemokine eotaxin into the media,relative to control, (p<0.05; FIG. 5), while the other treatments(theophylline alone, theophylline/leucine/resveratrol, leucineresveratrol, and TNFα) exerted no significant effect. As can be seenhere, the combined treatment of theophylline/leucine had a synergisticeffect of at least about 70% on eotaxin secretion, relative to thecontrol.

The effects shown in FIG. 5 are a direct effect on MLEC, as theconditioned media experiments produced no significant effect on eotaxin(FIG. 6).

Cellular expression of IL 6 was unaffected by theophylline, but thetheophylline/leucine and theophylline/leucine/resveratrol treatmentscaused modest, significant decreases (p<0.02; FIG. 7).

Similarly, IL 6 secretion into the media was decreased by ˜50% by boththe theophylline/leucine and theophylline/leucine/resveratrol treatments(p<0.0001; FIG. 8).

These direct effects were not recapitulated in the conditioned mediaexperiments (FIG. 9), demonstrating that this anti-inflammatory effectis not mediated by alterations in adipocyte cytokine production.

There was no direct effect of any of the treatments on MLEC expressionof either adiponectin receptor-1 (FIG. 10) or adiponectin receptor 2(FIG. 11).

However, treatment of adipocytes with thetheophylline/leucine/resveratrol combination produced conditioned mediathat, when applied to MLEC, elicited a marked increase in the expressionof the anti-inflammatory adiponectin-2 receptor in MLEC (p=0.0018, FIG.12). As can be seen in FIG. 12, only treatment withtheophylline/resveratrol/leucine yielded an improvement relative tocontrol, suggesting a synergistic effect between all three components.For this combination, the synergistic effect was about 100%.

Similarly, when the theophylline was replaced with theobromine, anothermethylxanthine/non-specific PDE inhibitor, the theobromine/leucine andleucine/resveratrol combinations stimulated modest, significantincreases in adiponectin receptor 1 (p<0.03, FIG. 13), with a further,more robust increase with the theobromine/leucine/resveratrolcombination (p<0.0005, FIG. 13).

Both the theobromine/leucine and theobromine/leucine/resveratrolcombinations stimulated robust increases in adiponectin receptor 2expression (p<0.0001, FIG. 14). As seen in FIG. 14, the combination oftheobromine with either leucine or leucine/resveratrol yielded asynergistic effect of about 100%.

These data demonstrate significant synergy between the non-specific PDEinhibitors theophylline and theobromine with leucine in attenuating theinflammatory responses that result in airway diseases, including asthmaand promoting anti-inflammatory responses. Notably, these effects areachieved at otherwise subtherapeutic doses of theophylline (1-2% oftherapeutically effective levels).

Example 2—Effects of Administration of Leucine and Theophylline to AdultFemale Balb/c Mice

Techniques

The interactive effects of leucine and theophylline was evaluated insixty young adult female Balb/c mice that were randomized to thefollowing six groups (n=10/group):

Group 1 was an untreated control group that was administered a dietcontaining a baseline level of leucine at 12 g of leucine/day.

Group 2 was a positive control group that was administered dexamethasonevia IP injection and a diet containing a baseline level of leucine at 12g of leucine/day.

Group 3 was administered a diet containing a high level of leucine (dietcontaining leucine at 12 g/day for days 0-13 and 24 g/day beginning onday 14) and no theophylline or dexamethasone.

Group 4 was administered sub-therapeutic level of theophylline (1 mg/kgtheophylline, 10% of therapeutic level) via IP injection and a highleucine diet (diet containing leucine at 12 g/day for days 0-13 and 24g/day beginning on day 14).

Group 5 was administered sub-therapeutic level of theophylline (3 mg/kgof theophylline, 30% of therapeutic level) via IP injection and a highleucine diet (diet containing leucine at 12 g/day for days 0-13 and 24g/day beginning on day 14).

Group 6 was administered a therapeutic level of theophylline group thatwas administered 10 mg/kg of theophylline (100% of therapeutic level)along with a diet containing a baseline of leucine at 12 g ofleucine/day.

The dosing and diet for each group is set out in further detail in thetable below.

Volume Group Group Dose Level Dosage Number Size Description Diet Route(mg/kg) (ml/kg) Dosing Regime 1 n = 10 Vehicle/ 12 g Leucine diet IP NA10 ml/kg Once daily on days Untreated 14-27 control 2 n = 10Dexamethasone/ 12 g Leucine diet IP 10 mg/kg 10 ml/kg Once daily on daysPositive control 25 to 27 3 n = 10 Vehicle/High 12 g Leucine diet IP NA10 ml/kg Once daily on days Leucine from day 0-13, 14-27 4 n = 10  10%thereafter switch to IP  1 mg/kg 10 ml/kg Theophylline 24 g Leucine diet  5 n = 10  30% on day 14 IP  3 mg/kg 10 ml/kg Theophylline 6 n = 10100% 12 g Leucine diet IP 10 mg/kg 10 ml/kg Theophylline/ BaselineLeucine

Study length was 28 days, and asthma was induced via ovalbumin (OVA)sensitization on days 0 and 14 via IP injection, followed by intranasalOVA challenge on days 14 and 25-27.

Animals were sacrificed on day 28, subjected to bronchial alveolarlavage and the lavage fluid (BALF, 2 mL) and lungs collected. Airwayinflammation was evaluated by assessing cellularity of BALF via flowcytometer; parameters used for cellular differentiation were sizedgranularity, autofluorescence and the expression of the followingmarkers: CD45, Gr-1, CD11b, CCR3, B220 and CD3.

Cell types were defined as follows:

Eosinophils: CD45⁺; Non-autofluorescent; CCR3⁺.

Neutrophils: CD45⁺; Non-autofluorescent; Gr-1⁺; CCR3⁺.

T cells: CD45⁺; Non-autofluorescent; Gr-1⁻; CD3⁺.

B cells: CD45⁺; Non-autofluorescent; Gr-1^(⋅); B220⁺.

Macrophages: CD45⁺, autofluorescent: CD11b⁺.

The non-cellular fraction of the BALF was analyzed for levels of IL-4,IL-5 and IL-13.

Results

Administration of the steroid (dexamethasone) significantly reduced BALFtotal cellularity as well as eosinophil, neutrophil, T-cell and B-cellnumber. Therapeutic levels of theophylline (10 mg/kg) resulted in modestsignificant decreases in eosinophil number, and the combination ofleucine with 3 mg/kg theophylline exerted a comparable effect, as shownin FIG. 15.

Both therapeutic theophylline and leucine combined with 3 mg/kgtheophylline modestly reduced T-cell number to a comparable degree, butthis trend did not reach significance for either treatment (0.05<p<0.1).The lowest level of theophylline (1 mg/kg) combined with theophyllineresulted in an increase in T-cells. These data are summarized in FIG.16.

However, the combination of leucine with subtherapeutic theophylline (3mg/kg) did significantly reduce the percentage of T cells in the BALF,as shown in FIG. 17.

The treatments exerted no significant effect on IL-4, IL-5 or IL-13.

These data indicate that leucine combined with a subtherapeutic dose oftheophylline attenuates the lymphocyte-mediated immune response inOVA-induced asthmatic mice.

It should be understood from the foregoing that, while particularimplementations have been illustrated and described, variousmodifications can be made thereto and are contemplated herein. It isalso not intended that the invention be limited by the specific examplesprovided within the specification. While the invention has beendescribed with reference to the aforementioned specification, thedescriptions and illustrations of the preferable embodiments herein arenot meant to be construed in a limiting sense. Furthermore, it shall beunderstood that all aspects of the invention are not limited to thespecific depictions, configurations or relative proportions set forthherein which depend upon a variety of conditions and variables. Variousmodifications in form and detail of the embodiments of the inventionwill be apparent to a person skilled in the art. It is thereforecontemplated that the invention shall also cover any such modifications,variations and equivalents.

What is claimed is:
 1. A composition comprising: (a) leucine and/or oneor more leucine metabolites selected from the group consisting ofketo-isocaproic acid (KIC), alpha-hydroxy-isocaproic acid, andhydroxymethylbutyrate (HMB); and (b) a methylxanthine; wherein thecomposition comprises at least 250 mg of leucine and/or between 10-200mg of the one or more leucine metabolites; wherein the compositioncomprises between 1-80 mg of the methylxanthine; wherein the compositionconsists of components (a) and (b), optionally resveratrol, andoptionally pharmaceutically acceptable excipients; wherein thecomposition is formulated as a tablet, capsule or gel capsule, andwherein the components (a) and (b) are administered simultaneously or inclose temporal proximity.
 2. The composition claim 1, wherein thecomposition comprises leucine.
 3. The composition of claim 1, whereinthe composition comprises at least 500 mg of leucine.
 4. The compositionof claim 1, wherein the composition comprises between 250-1500 mg ofleucine.
 5. The composition of claim 1, wherein the compositioncomprises between 10-100 mg of leucine metabolites.
 6. The compositionof claim 1, wherein the composition comprises between 100-200 mg ofleucine metabolites.
 7. The composition of claim 1, wherein themethylxanthine is selected from the group consisting of theophylline andtheobromine.
 8. The composition of claim 7, wherein the compositioncomprises at least about 5 mg of theophylline.
 9. The composition ofclaim 7, wherein the composition comprises between about 1-25 mg oftheophylline.
 10. The composition of claim 7, wherein the compositioncomprises a sub-therapeutic amount of theophylline.
 11. The compositionof claim 1, wherein the composition comprises a sub-therapeutic amountof the methylxanthine.
 12. The composition of claim 1, wherein thecomposition is substantially free of non-branched amino acids.
 13. Thecomposition of claim 1, wherein the percent of non-branched amino acidsrelative to total amino acids in the composition is less than about 10%.14. The composition of claim 1, further comprising resveratrol.
 15. Thecomposition of claim 14, wherein the composition comprises at least 35mg of resveratrol.
 16. The composition of claim 14, wherein thecomposition comprises between 5-500 mg of resveratrol.
 17. Thecomposition of claim 1, wherein the composition further comprisespharmaceutically acceptable excipients.
 18. The composition of claim 1,wherein the composition further comprises an anti-diabetic agent. 19.The composition of claim 1, wherein the composition further comprises asirtuin pathway activator.
 20. The composition of claim 1, wherein thecomposition is formulated as a unit dosage.
 21. The composition of claim1, wherein the composition is formulated for sustained release to effecta circulating level of the methylxanthine at greater than about 1 μMover a time period of at least about 4 hours.
 22. The composition ofclaim 1, wherein the mass ratio of (a) to (b) is at least about 15, andwherein the composition comprises at least about 5 mg of themethylxanthine.
 23. The composition of claim 22, further comprising atleast about 10 mg of resveratrol.
 24. The composition of claim 22,wherein the methylxanthine is theophylline or theobromine.
 25. A kitcomprising a multi-day supply of unit dosages of the composition ofclaim 1 and instructions directing the administration of said multi-daysupply over a period of multiple days.
 26. A method of treating chronicobstructive pulmonary disease (DOPD) or asthma in a subject in need oftreatment comprising administering to the subject a composition ofclaim
 1. 27. The method of claim 26, wherein the subject is a human, adomesticated animal, or a farm animal.
 28. A method of increasingexpression level or secretion of an inflammatory marker selected fromthe group consisting of NFκB, eotaxin, IL1-β, and IL6, or reducingexpression level or secretion of an anti-inflammatory marker selectedfrom the group consisting of adiponectin receptor 1 and adiponectinreceptor 2, the method comprising contacting a lung endothelial cellwith a composition of claim 1 to effect said reduction and/or increasein expression level or secretion of said inflammatory or saidanti-inflammatory marker.